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1.  PRKCA and Multiple Sclerosis: Association in Two Independent Populations 
PLoS Genetics  2006;2(3):e42.
Multiple sclerosis (MS) is a chronic disease of the central nervous system responsible for a large portion of neurological disabilities in young adults. Similar to what occurs in numerous complex diseases, both unknown environmental factors and genetic predisposition are required to generate MS. We ascertained a set of 63 Finnish MS families, originating from a high-risk region of the country, to identify a susceptibility gene within the previously established 3.4-Mb region on 17q24. Initial single nucleotide polymorphism (SNP)-based association implicated PRKCA (protein kinase C alpha) gene, and this association was replicated in an independent set of 148 Finnish MS families (p = 0.0004; remaining significant after correction for multiple testing). Further, a dense set of 211 SNPs evenly covering the PRKCA gene and the flanking regions was selected from the dbSNP database and analyzed in two large, independent MS cohorts: in 211 Finnish and 554 Canadian MS families. A multipoint SNP analysis indicated linkage to PRKCA and its telomeric flanking region in both populations, and SNP haplotype and genotype combination analyses revealed an allelic variant of PRKCA, which covers the region between introns 3 and 8, to be over-represented in Finnish MS cases (odds ratio = 1.34, 95% confidence interval 1.07–1.68). A second allelic variant, covering the same region of the PRKCA gene, showed somewhat stronger evidence for association in the Canadian families (odds ratio = 1.64, 95% confidence interval 1.39–1.94). Initial functional relevance for disease predisposition was suggested by the expression analysis: The transcript levels of PRKCA showed correlation with the copy number of the Finnish and Canadian “risk” haplotypes in CD4-negative mononuclear cells of five Finnish multiplex families and in lymphoblast cell lines of 11 Centre d'Etude du Polymorphisme Humain (CEPH) individuals of European origin.
Complex diseases such as multiple sclerosis (MS) likely result from problems in networks of interactions between several genes and largely unidentified environmental and lifestyle factors. Identification of MS-specific genes has been challenging. HLA-DRB1*15 is the only consistent locus observed in most populations; however, the recent genome scan on more than 700 European families implicated 17q as a second-best MS locus [12]. Since MS families from the high-risk region of Finland initially revealed linkage to 17q, the authors used the regionally ascertained set of 63 families to identify a MS predisposing gene within a major non–HLA locus on 17q. The initial association was observed with single nucleotide polymorphisms (SNPs) located in intron 3 of the PRKCA (protein kinase C alpha) gene in Finnish MS families and replicated in an independent set of 148 MS families from Finland and 554 from Canada, two populations with a different genetic background. Combining the data of two SNP variants revealed two allele combinations of PRKCA, which were over-represented in Finnish or Canadian MS cases (odds ratio = 1.34, 95% confidence interval, 1.07–1.68, and odds ratio = 1.64, 95% confidence interval 1.39–1.94, respectively). Linkage and association of the PRKCA gene, encoding a regulator of immune responses, in two populations imply its involvement in the etiology of MS.
PMCID: PMC1420678  PMID: 16596167
2.  Characterisation of SNP haplotype structure in chemokine and chemokine receptor genes using CEPH pedigrees and statistical estimation 
Human Genomics  2004;1(3):195-207.
Chemokine signals and their cell-surface receptors are important modulators of HIV-1 disease and cancer. To aid future case/control association studies, aim to further characterise the haplotype structure of variation in chemokine and chemokine receptor genes. To perform haplotype analysis in a population-based association study, haplotypes must be determined by estimation, in the absence of family information or laboratory methods to establish phase. Here, test the accuracy of estimates of haplotype frequency and linkage disequilibrium by comparing estimated haplotypes generated with the expectation maximisation (EM) algorithm to haplotypes determined from Centre d'Etude Polymorphisme Humain (CEPH) pedigree data. To do this, they have characterised haplotypes comprising alleles at 11 biallelic loci in four chemokine receptor genes (CCR3, CCR2, CCR5 and CCRL2), which span 150 kb on chromosome 3p21, and haplotyes of nine biallelic loci in six chemokine genes [MCP-1(CCL2), Eotaxin(CCL11), RANTES(CCL5), MPIF-1(CCL23), PARC(CCL18) and MIP-1α(CCL3) ] on chromosome 17q11-12. Forty multi-generation CEPH families, totalling 489 individuals, were genotyped by the TaqMan 5'-nuclease assay. Phased haplotypes and haplotypes estimated from unphased genotypes were compared in 103 grandparents who were assumed to have mated at random.
For the 3p21 single nucleotide polymorphism (SNP) data, haplotypes determined by pedigree analysis and haplotypes generated by the EM algorithm were nearly identical. Linkage disequilibrium, measured by the D' statistic, was nearly maximal across the 150 kb region, with complete disequilibrium maintained at the extremes between CCR3-Y17Y and CCRL2-1243V. D'-values calculated from estimated haplotypes on 3p21 had high concordance with pairwise comparisons between pedigree-phased chromosomes. Conversely, there was less agreement between analyses of haplotype frequencies and linkage disequilibrium using estimated haplotypes when compared with pedigree-phased haplotypes of SNPs on chromosome 17q11-12. These results suggest that, while estimations of haplotype frequency and linkage disequilibrium may be relatively simple in the 3p21 chemokine receptor cluster in population samples, the more complex environment on chromosome 17q11-12 will require a higher resolution haplotype analysis.
PMCID: PMC3525080  PMID: 15588479
chemokine; SNP; haplotype estimation; pedigree analysis; linkage disequilibrium
3.  An Evaluation of the Performance of Tag SNPs Derived from HapMap in a Caucasian Population 
PLoS Genetics  2006;2(3):e27.
The Haplotype Map (HapMap) project recently generated genotype data for more than 1 million single-nucleotide polymorphisms (SNPs) in four population samples. The main application of the data is in the selection of tag single-nucleotide polymorphisms (tSNPs) to use in association studies. The usefulness of this selection process needs to be verified in populations outside those used for the HapMap project. In addition, it is not known how well the data represent the general population, as only 90–120 chromosomes were used for each population and since the genotyped SNPs were selected so as to have high frequencies. In this study, we analyzed more than 1,000 individuals from Estonia. The population of this northern European country has been influenced by many different waves of migrations from Europe and Russia. We genotyped 1,536 randomly selected SNPs from two 500-kbp ENCODE regions on Chromosome 2. We observed that the tSNPs selected from the CEPH (Centre d'Etude du Polymorphisme Humain) from Utah (CEU) HapMap samples (derived from US residents with northern and western European ancestry) captured most of the variation in the Estonia sample. (Between 90% and 95% of the SNPs with a minor allele frequency of more than 5% have an r2 of at least 0.8 with one of the CEU tSNPs.) Using the reverse approach, tags selected from the Estonia sample could almost equally well describe the CEU sample. Finally, we observed that the sample size, the allelic frequency, and the SNP density in the dataset used to select the tags each have important effects on the tagging performance. Overall, our study supports the use of HapMap data in other Caucasian populations, but the SNP density and the bias towards high-frequency SNPs have to be taken into account when designing association studies.
The recent completion of the Haplotype Map (HapMap) project of the human genome provides considerable information on the patterns of variation in the genome of four populations. One of the applications is a description of a set of tags that act as proxies for many other surrounding variants. This will greatly help researchers in their quest to find complex disease genes by reducing the number of genetic variants to test in association studies. To evaluate its usefulness, several aspects of the map, including its transferability to other populations, still needed to be verified experimentally. Using genomic regions where variants had been thoroughly documented in Caucasian samples from Estonia, the researchers found that the transferability of tags is extremely good. The researchers also found that variants with low frequency in the general population (i.e., less than 5%) could not be accurately captured with tags, and that the regional density of variants in the HapMap project had a major impact on the performance of the tags. This research indicates that the HapMap project will be useful, but that careful consideration of hypotheses and study design will be essential for the success of association studies.
PMCID: PMC1391920  PMID: 16532062
4.  Association of the HLA locus and TNF with type I autoimmune hepatitis susceptibility in New Zealand Caucasians 
SpringerPlus  2013;2:355.
The precise etiology of autoimmune hepatitis (AIH) remains unknown, although a number of genetic loci have been implicated in the susceptibility of type 1 AIH. The purpose of this study was to test for association of these loci with type 1 AIH in New Zealand Caucasians.
77 AIH patients and 485 healthy controls were genotyped for the SNPs rs2187668 (HLA-DRB*03:01), rs660895 (HLA-DRB*04:01), rs3749971 (HLA-A1-B8-DR3), rs231775 (CLTLA4), rs1800629 (TNF), and rs1800682 (FAS) using predesigned TaqMan SNP genotyping assays. Chi square analysis was used to test for association of allele and genotype with overall AIH, and with severe fibrosis and ALT levels at 6 months.
Significant risk of AIH was conferred by the minor alleles of rs2187668 (OR = 2.45, 95% CI 1.65-3.61, p < 0.0001), rs3749971 (OR = 1.89, 95% CI 1.21-2.94, p = 0.004) and rs1800629 (OR = 2.06, 95% CI 1.41-3.01, p = 0.0001). Multivariate analysis showed that rs2187668 was independently associated with type 1 AIH susceptibility (OR = 2.40, 95% CI 1.46-3.93, p = 0.001). The C allele of FAS SNP rs1800682 was associated with increased risk of severe fibrosis at diagnosis (OR = 2.03, 95% CI 1.05-3.93, p = 0.035) and with incomplete normalization of ALT levels at 6 months post-diagnosis (OR = 3.94, 95% CI 1.62-9.54, p = 0.0015).
This is the first population-based study to investigate genetic risk loci for type 1 AIH in New Zealand Caucasians. We report significant independent association of HLA-DRB1*03:01 with overall susceptibility to type 1 AIH, as well as FAS with a more aggressive disease phenotype.
PMCID: PMC3733077  PMID: 23961418
HLA-DRB*03:01; HLA-DRB*04:01; HLA-A1-B8-DR3; FAS; CTLA4; Severe fibrosis; ALT levels
5.  Sex differences in TTC12/ANKK1 haplotype associations with daily tobacco smoking in Black and White Americans 
Nicotine & Tobacco Research  2010;12(3):251-262.
The 11q23.1 genomic region has been associated with nicotine dependence in Black and White Americans.
By conducting linkage disequilibrium analyses of 7 informative single nucleotide polymorphisms (SNPs) within the tetratricopeptide repeat domain 12 (TTC12)/ankyrin repeat and kinase containing 1 (ANKK1)/dopamine (D2) receptor gene cluster, we identified haplotype block structures in 270 Black and 368 White (n = 638) participants, from the Baltimore Epidemiologic Catchment Area cohort study, spanning the TTC12 and ANKK1 genes consisting of three SNPs (rs2303380–rs4938015–rs11604671). Informative haplotypes were examined for sex-specific associations with daily tobacco smoking initiation and cessation using longitudinal data from 1993–1994 and 2004–2005 interviews.
There was a Haplotype × Sex interaction such that Black men possessing the GTG haplotype who were smokers in 1993–2004 were more likely to have stopped smoking by 2004–2005 (55.6% GTG vs. 22.0% other haplotypes), while Black women were less likely to have quit smoking if they possessed the GTG (20.8%) versus other haplotypes (24.0%; p = .028). In Whites, the GTG haplotype (vs. other haplotypes) was associated with lifetime history of daily smoking (smoking initiation; odds ratio = 1.6; 95% CI = 1.1–2.4; p = .013). Moreover, there was a Haplotype × Sex interaction such that there was higher prevalence of smoking initiation with GTG (77.6%) versus other haplotypes (57.0%; p = .043).
In 2 different ethnic American populations, we observed man–woman variation in the influence of the rs2303380–rs4938015–rs11604671 GTG haplotype on smoking initiation and cessation. These results should be replicated in larger cohorts to establish the relationship among the rs2303380–rs4938015–rs11604671 haplotype block, sex, and smoking behavior.
PMCID: PMC2825103  PMID: 20133381
6.  Marked differences in frequencies of statin therapy relevant SLCO1B1 variants and haplotypes between Roma and Hungarian populations 
BMC Genetics  2015;16:108.
SLCO1B1 polymorphisms are relevant in statin pharmacokinetics. Aim of this study was to investigate the genetic variability and haplotype profile of SLCO1B1 polymorphisms in Roma and Hungarian populations. Genotypes of 470 Roma and 442 Hungarian subjects for c.388A > G, c.521T > C and c.1498-1331T > C polymorphisms were determined by PCR-RFLP assay. Using these SNPs eight different haplotypes could be differentiated.
Differences were found between Roma and Hungarians in SLCO1B1 388AA (24.5 vs. 45.5 %), GG (33.4 vs. 17.9 %) genotypes, AG + GG (75.5 vs. 54.5 %) carriers, in G allele frequency (0.545 vs. 0.362), respectively (p < 0.001). The most common SLCO1B1 haplotype was the ht8 (GTT) both in Roma (43.6 %) and in Hungarian (59.1 %) samples. The ht6 (GCT) was not present in Roma population samples Haplotype analyses showed striking differences between the Roma and Hungarian samples in ht4 (ATT, 37.2 % vs 20.8 %), ht5 (GCC, 1.15 % vs. 3.62 %) and ht8 (GTT, 43.6 % vs. 59.1 %) haplotypes (p < 0.01), respectively. Linkage disequilibrium analysis showed that the studied variants are in different linkage disequilibrium patterns depending on the ethnic origin.
Similarly to Caucasians the 388G is the minor allele in Hungarians, however, in Roma the 388A was found to be the minor allele contrary to Indians (India). The minor allele frequency of 521T > C and 1498-1331T > C SNPs are almost three times higher in Romas than in Indians (Singapore and Gujarati, respectively). Observed allele frequency for 1498-1331T > C polymorphism reflects the measured average European rates in Hungarians. The results can be applied in population specific treatment algorithms when developing effective programs for statin therapy.
PMCID: PMC4559300  PMID: 26334733
SLCO1B1; Statin; Haplotype; Roma; Hungarian; Pharmacogenetics
7.  Genetic Linkage and Association of the Growth Hormone Secretagogue Receptor (Ghrelin Receptor) Gene in Human Obesity 
Diabetes  2005;54(1):259-267.
The growth hormone secretagogue receptor (GHSR) (ghrelin receptor) plays an important role in the regulation of food intake and energy homeostasis. The GHSR gene lies on human chromosome 3q26 within a quantitative trait locus strongly linked to multiple phenotypes related to obesity and the metabolic syndrome. Because the biological function and location of the GHSR gene make it an excellent candidate gene, we tested the relation between common single nucleotide polymorphisms (SNPs) in the GHSR gene and human obesity. We performed a comprehensive analysis of SNPs, linkage disequilibrium (LD), and haplotype structure across the entire GHSR gene region (99.3 kb) in 178 pedigrees with multiple obese members (DNA of 1,095 Caucasians) and in an independent sample of the general population (MONICA Augsburg left ventricular hypertrophy substudy; DNA of 1,418 Caucasians). The LD analysis revealed a disequilibrium block consisting of five SNPs, consistent in both study cohorts. We found linkage among all five SNPs, their haplotypes, and BMI. Further, we found suggestive evidence for transmission disequilibrium for the minor SNP alleles (P < 0.05) and the two most common haplotypes with the obesity affection status (“susceptible” P = 0.025, “nonsusceptible” P = 0.045) in the family cohort using the family-based association test program. Replication of these findings in the general population resulted in stronger evidence for an association of the SNPs (best P = 0.00001) and haplotypes with the disease (“susceptible” P = 0.002, “nonsusceptible” P = 0.002). To our knowledge, these data are the first to demonstrate linkage and association of SNPs and haplotypes within the GHSR gene region and human obesity. This linkage, together with significant transmission disequilibrium in families and replication of this association in an independent population, provides evidence that common SNPs and haplotypes within the GHSR region are involved in the pathogenesis of human obesity.
PMCID: PMC2793077  PMID: 15616037
8.  HLA-G UTR Haplotype Conservation in the Malian Population: Association with Soluble HLA-G 
PLoS ONE  2013;8(12):e82517.
The HLA-G molecule plays an important role in immunomodulation. In a previous study carried out on a southern French population our team showed that HLA-G haplotypes, defined by SNPs in the coding region and specific SNPs located in 5′URR and 3′UTR regulatory regions, are associated with differential soluble HLA-G expression (sHLA-G). Furthermore, the structure of these HLA-G haplotypes appears to be conserved in geographically distant populations.
The aim of our study is to confirm these expectations in a sub-Saharan African population and to explore additional factors, such as HLA-A alleles, that might influence sHLA-G expression.
DNA and plasma samples were collected from 229 Malians; HLA-G and HLA-A genotyping were respectively performed by the Snap Shot® method and by Luminex™ technology. sHLA-G dosage was performed using an ELISA kit. HLA-G and HLA-A allelic and haplotypic frequencies were estimated using an EM algorithm from the Gene[Rate] program. Associations between genetic and non genetic parameters with sHLA-G were performed using a non-parametric test with GRAPH PAD Prism 5.
Our results reveal a good conservation of the HLA-G UTR haplotype structure in populations with different origins and demographic histories. These UTR haplotypes appear to be involved in different sHLA-G expression patterns. Specifically, the UTR-2 haplotype was associated with low sHLA-G levels, displaying a dominant negative effect. Furthermore, an allelic effect of both HLA-G and HLA-A, as well as non genetic parameters, such as age and gender possibly linked to osteogenesis and sexual hormones, also seem to be involved in the modulation of sHLA-G.
These data suggest that further investigation in larger cohorts and in populations from various ethnical backgrounds is necessary not only to detect new functional polymorphism in HLA-G regulatory regions, but also to reveal the extent of biological phenomena that influence sHLA-G secretion and this might therefore have an impact on transplantation practice.
PMCID: PMC3871591  PMID: 24376542
9.  MHC Haplotype Matching for Unrelated Hematopoietic Cell Transplantation 
PLoS Medicine  2007;4(1):e8.
Current criteria for the selection of unrelated donors for hematopoietic cell transplantation (HCT) include matching for the alleles of each human leukocyte antigen (HLA) locus within the major histocompatibility complex (MHC). Graft-versus-host disease (GVHD), however, remains a significant and potentially life-threatening complication even after HLA-identical unrelated HCT. The MHC harbors more than 400 genes, but the total number of transplantation antigens is unknown. Genes that influence transplantation outcome could be identified by using linkage disequilibrium (LD)-mapping approaches, if the extended MHC haplotypes of the unrelated donor and recipient could be defined.
Methods and Findings
We isolated DNA strands extending across 2 million base pairs of the MHC to determine the physical linkage of HLA-A, -B, and -DRB1 alleles in 246 HCT recipients and their HLA-A, -B, -C, -DRB1, -DQB1 allele-matched unrelated donors. MHC haplotype mismatching was associated with a statistically significantly increased risk of severe acute GVHD (odds ratio 4.51; 95% confidence interval [CI], 2.34–8.70, p < 0.0001) and with lower risk of disease recurrence (hazard ratio 0.45; 95% CI, 0.22–0.92, p = 0.03).
The MHC harbors genes that encode unidentified transplantation antigens. The three-locus HLA-A, -B, -DRB1 haplotype serves as a proxy for GVHD risk among HLA-identical transplant recipients. The phasing method provides an approach for mapping novel MHC-linked transplantation determinants and a means to decrease GVHD-related morbidity after HCT from unrelated donors.
A novel method of MHC haplotype matching provides a means to decrease graft-versus-host disease-related morbidity after transplantation from unrelated donors.
Editors' Summary
Graft rejection and graft-versus-host disease (GVHD) are feared complications of hematopoietic cell transplantation (HCT). GVHD can affect all parts of the body, and, if severe (grade III to IV out of a scale of IV), can lead to the death of the transplant recipient. GVHD or rejection of the graft occurs when there are differences in specific proteins involved in the immune response (known as HLA antigens) between donor and recipient that stimulate the immune reaction. GVHD and graft rejection occur most often in people who receive transplants from unrelated donors because, although when donors are matched to recipients matching is done for the most important HLA antigens known to be involved, it has not technically been possible to match for all possible antigens. However, the human genome is organized into segments or blocks of closely linked genetic variants that are inherited as “haplotypes” on the same DNA strand of a chromosome. Most of the genes that code for HLA antigens are physically located together in one part of the human genome, known as the MHC region. Currently three HLA markers from this region (HLA-A, -B, -DRB1) are matched when matching donors and recipients. If it were possible to better map the structure of this region, it would be possible to better match recipients and donors (especially unrelated donors) for the unidentified transplantation antigens and reduce the chance of recipients getting GVHD or rejecting their grafts.
Why Was This Study Done?
Current strategies to define MHC haplotype blocks look at, on average, a length of only 18,000 base pairs and hence cannot define extended MHC haplotypes. Previously, this group of researchers developed a method of defining the HLA-A, B, DR haplotypes in recipients and their HLA-matched unrelated transplant donors using high-quality DNA containing 2 million base pairs across the MHC region. They wanted see if using this technique might provide a way to better assess the risk recipients have of developing GVHD or of having recurrent disease.
What Did the Researchers Do and Find?
They studied 246 HCT recipients and their donors who had been matched for HLA-A, -B, -C, -DRB1, -DQB1 by current techniques. The recipients were having HCT for a variety of hematological cancers: acute lymphoid leukemia, acute myeloid leukemia, chronic myeloid leukemia, or myelodysplastic syndrome. They found that, using the new technique, 22% of the donor–recipient pairs were haplotype-mismatched. Taking various other factors into account, including age, and patient and donor gender, MHC haplotype mismatching was associated with an approximately four times greater risk of severe acute GVHD but with a lower risk of disease recurrence. The lower risk of recurrence is believed to be because transplanted cells do not only replace abnormal cancerous cells but also react against them and therefore decrease the chance of the cancer recurring; mismatched cells are known to be more stimulated to react against the cancerous cells.
What Do These Findings Mean?
The results here suggest that this new haplotype matching method can provide a way to assess the risk of GVHD after HCT from unrelated donors, and in future could be considered as a technique to match donors and recipients.
Additional Information.
Please access these Web sites via the online version of this summary at
• Medline Plus has a page of information on stem cell transplantation, including HCT
• The Anthony Nolan Trust holds one of the largest databases of unrelated donors in the world
• The National Cancer Institute has a page of questions and answers on HCT
• The Center for International Blood & Marrow Transplant Research describes outcomes research in transplantation
• The National Marrow Donor Program describes how HLA-typed unrelated donors are identified
• The World Marrow Donor Association is involved in facilitating stem cell donation across international boundaries
PMCID: PMC1796628  PMID: 17378697
10.  Fine Mapping of the Psoriasis Susceptibility Locus PSORS1 Supports HLA-C as the Susceptibility Gene in the Han Chinese Population 
PLoS Genetics  2008;4(3):e1000038.
PSORS1 (psoriasis susceptibility gene 1) is a major susceptibility locus for psoriasis. Several fine-mapping studies have highlighted a 300-kb candidate region of PSORS1 where multiple biologically plausible candidate genes were suggested. The most recent study has indicated HLA-Cw6 as the primary PSORS1 risk allele within the candidate region in a Caucasian population. In this study, a family-based association analysis of the PSORS1 locus was performed by analyzing 10 polymorphic microsatellite markers from the PSORS1 region as well as HLA-B, HLA-C and CDSN loci in 163 Chinese families of psoriasis. Five marker loci show strong evidence (P<10−3), and one marker locus shows weak evidence (P = 0.04) for association. The haplotype cluster analysis showed that all the risk haplotypes are Cw6 positive and share a 369-kb region of homologous marker alleles which carries all the risk alleles, including HLA-Cw6 and CDSN*TTC, identified in this study. The recombinant haplotype analysis of the HLA-Cw6 and CDSN*TTC alleles in 228 Chinese families showed that the HLA-Cw6−/CDSN*TTC+ recombinant haplotype is clearly not associated with risk for psoriasis (T∶NT = 29:57, p = 0.0025) in a Chinese population, suggesting that the CDSN*TTC allele itself does not confer risk without the presence of the HLA-Cw6 allele. The further exclusion analysis of the non-risk HLA-Cw6−/CDSN*TTC+ recombinant haplotypes with common recombination breakpoints has allowed us to refine the location of PSORS1 to a small candidate region. Finally, we performed a conditional linkage analysis and showed that the HLA-Cw6 is a major risk allele but does not explain the full linkage evidence of the PSORS1 locus in a Chinese population. By performing a series of family-based association analyses of haplotypes as well as an exclusion analysis of recombinant haplotypes, we were able to refine the PSORS1 gene to a small critical region where HLA-C is a strong candidate to be the PSORS1 susceptibility gene.
Author Summary
Psoriasis is a common skin disease with strong genetic risk. The analysis of psoriatic families with multiple affected individuals has identified several genomic regions that are linked (showing linkage evidence) to the development of psoriasis. Of them, the region on 6p21.3 (PSORS1) is a well-confirmed major risk locus. The identification of the disease risk gene within the PSOR1 locus, however, has been difficult, largely due to the fact that several genes show evidence for association with the disease development and the evidences are highly correlated and hard to be separated from each other. In this study, we performed a fine mapping study of the PSORS1 locus in Chinese families with psoriasis. By analyzing recombinant haplotypes that carry different risk-associated genetic variants within different genes, we were able to separate the genetic effects observed within multiple genes and identify strong supporting evidence for HLA-C to be the primary risk gene of the PSORS1 locus. We have further demonstrated that the genetic variation within the HLA-C gene does not explain the full linkage evidence at the PSORS1 locus, suggesting that there might be other risk genes and/or alleles within the region. Our findings have improved our understanding about the genetic complexity of the PSORS1 locus.
PMCID: PMC2265413  PMID: 18369457
11.  HIV Control through a Single Nucleotide on the HLA-B Locus 
Journal of Virology  2012;86(21):11493-11500.
Genetic variation within the HLA-B locus has the strongest impact on HIV disease progression of any polymorphisms within the human genome. However, identifying the exact mechanism involved is complicated by several factors. HLA-Bw4 alleles provide ligands for NK cells and for CD8 T cells, and strong linkage disequilibrium between HLA class I alleles complicates the discrimination of individual HLA allelic effects from those of other HLA and non-HLA alleles on the same haplotype. Here, we exploit an experiment of nature involving two recently diverged HLA alleles, HLA-B*42:01 and HLA-B*42:02, which differ by only a single amino acid. Crucially, they occur primarily on identical HLA class I haplotypes and, as Bw6 alleles, do not act as NK cell ligands and are therefore largely unconfounded by other genetic factors. We show that in an outbred cohort (n = 2,093) of HIV C-clade-infected individuals, a single amino acid change at position 9 of the HLA-B molecule critically affects peptide binding and significantly alters the cytotoxic T lymphocyte (CTL) epitopes targeted, measured directly ex vivo by gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay (P = 2 × 10−10) and functionally through CTL escape mutation (P = 2 × 10−8). HLA-B*42:01, which presents multiple Gag epitopes, is associated with a 0.52 log10 lower viral-load set point than HLA-B*42:02 (P = 0.02), which presents no p24 Gag epitopes. The magnitude of this effect from a single amino acid difference in the HLA-A*30:01/B*42/Cw*17:01 haplotype is equivalent to 75% of that of HLA-B*57:03, the most protective HLA class I allele in this population. This naturally controlled experiment represents perhaps the clearest demonstration of the direct impact of a particular HIV-specific CTL on disease control.
PMCID: PMC3486337  PMID: 22896606
12.  Identification of a Polymorphic Gene, BCL2A1, Encoding Two Novel Hematopoietic Lineage-specific Minor Histocompatibility Antigens 
The Journal of Experimental Medicine  2003;197(11):1489-1500.
We report the identification of two novel minor histocompatibility antigens (mHAgs), encoded by two separate single nucleotide polymorphisms on a single gene, BCL2A1, and restricted by human histocompatibility leukocyte antigen (HLA)-A*2402 (the most common HLA-A allele in Japanese) and B*4403, respectively. Two cytotoxic T lymphocyte (CTL) clones specific for these mHAgs were first isolated from two distinct recipients after hematopoietic cell transplantation. Both clones lyse only normal and malignant cells within the hematopoietic lineage. To localize the gene encoding the mHAgs, two-point linkage analysis was performed on the CTL lytic patterns of restricting HLA-transfected B lymphoblastoid cell lines obtained from Centre d'Etude du Polymorphisme Humain. Both CTL clones showed a completely identical lytic pattern for 4 pedigrees and the gene was localized within a 3.6-cM interval of 15q24.3–25.1 region that encodes at least 46 genes. Of those, only BCL2A1 has been reported to be expressed in hematopoietic cells and possess three nonsynonymous nucleotide changes. Minigene transfection and epitope reconstitution assays with synthetic peptides identified both HLA-A*2402– and B*4403-restricted mHAg epitopes to be encoded by distinct polymorphisms within BCL2A1.
PMCID: PMC2193899  PMID: 12771180
minor histocompatibility antigen; hematopoietic cell transplantation; cytotoxic T lymphocyte; graft-versus-leukemia effect; linkage analysis
13.  Missingness in the T1DGC MHC fine-mapping SNP data: association with HLA genotype and potential influence on genetic association studies 
Diabetes, obesity & metabolism  2009;11(Suppl 1):101-107.
The absence or ‘missingness’ of single nucleotide polymorphism (SNP) assay values because of genotype or related factors of interest may bias association and other studies. Missingness was determined for the Type 1 Diabetes Genetics Consortium (T1DGC) Major Histocompatibility Complex (MHC) data and was found to vary across the region, ranging up to 11.1% of the non-null proband SNPs, with a median of 0.3%. We consider factors related to missingness in the T1DGC data and briefly assess its possible influence on association studies.
We assessed associations of missingness in the SNP assay data with human leucocyte antigen (HLA) genotype of the individual and with SNP genotypes of the parents. Within-cohort analyses were combined (over all cohorts) using (i) Mantel–Haenszel tests for two-by-two tables or (ii) by combining test statistics for larger tables and regression models. Mixed effect regression models were used to assess association of the SNP genotypes with affected status of the offspring after adjustment for parental SNP genotypes, cohort membership and HLA genotypes. Log-linear models were used to assess association of missingness in the unaffected sib assays with SNP genotypes of the probands.
Missingness of SNP values near the HLA class I (A, B and C) and class II (DR, DQ and DP) loci is strongly associated with carriage of corresponding HLA genotypes within these groups. Similar associations pertain to missing values among the microsatellite data. In at least some of these cases, regions of missingness coincided with known deletion regions corresponding to the associated HLA haplotype. We conjecture that other regions of associated missingness may point to similar haplotypic deletions. Analysis of association patterns of SNP genotypes with affected status of offspring does not indicate strong informative missingness. However, association of missingness in proband data with parental SNP genotypes may impact transmission disequilibrium test (TDT)-type analyses. Comparisons of affected and unaffected siblings point to possible susceptibility regions additional to the classical HLA-DR3/4 alleles near BAT4-LY6G5B-BAT5 and NOTCH4.
Potentially informative missingness in SNP assay values in the MHC region may impact on association and related analyses based on the T1DGC data. These results suggest that it would be prudent to assess the degree to which missingness may abrogate assessed SNP disease markers in such studies. Initial analyses based on comparison of affected and unaffected status in offspring suggest that at least these may be little affected.
PMCID: PMC2755067  PMID: 19143822
association studies; deletions; HLA; informative missingness; MHC fine mapping; type 1 diabetes
14.  Identification of improved IL28B SNPs and haplotypes for prediction of drug response in treatment of hepatitis C using massively parallel sequencing in a cross-sectional European cohort 
Genome Medicine  2011;3(8):57.
The hepatitis C virus (HCV) infects nearly 3% of the World's population, causing severe liver disease in many. Standard of care therapy is currently pegylated interferon alpha and ribavirin (PegIFN/R), which is effective in less than half of those infected with the most common viral genotype. Two IL28B single nucleotide polymorphisms (SNPs), rs8099917 and rs12979860, predict response to (PegIFN/R) therapy in treatment of HCV infection. These SNPs were identified in genome wide analyses using Illumina genotyping chips. In people of European ancestry, there are 6 common (more than 1%) haplotypes for IL28B, one tagged by the rs8099917 minor allele, four tagged by rs12979860.
We used massively parallel sequencing of the IL28B and IL28A gene regions generated by polymerase chain reaction (PCR) from pooled DNA samples from 100 responders and 99 non-responders to therapy, to identify common variants. Variants that had high odds ratios and were validated were then genotyped in a cohort of 905 responders and non-responders. Their predictive power was assessed, alone and in combination with HLA-C.
Only SNPs in the IL28B linkage disequilibrium block predicted drug response. Eighteen SNPs were identified with evidence for association with drug response, and with a high degree of confidence in the sequence call. We found that two SNPs, rs4803221 (homozygote minor allele positive predictive value (PPV) of 77%) and rs7248668 (PPV 78%), predicted failure to respond better than the current best, rs8099917 (PPV 73%) and rs12979860 (PPV 68%) in this cross-sectional cohort. The best SNPs tagged a single common haplotype, haplotype 2. Genotypes predicted lack of response better than alleles. However, combination of IL28B haplotype 2 carrier status with the HLA-C C2C2 genotype, which has previously been reported to improve prediction in combination with IL28B, provides the highest PPV (80%). The haplotypes present alternative putative transcription factor binding and methylation sites.
Massively parallel sequencing allowed identification and comparison of the best common SNPs for identifying treatment failure in therapy for HCV. SNPs tagging a single haplotype have the highest PPV, especially in combination with HLA-C. The functional basis for the association may be due to altered regulation of the gene. These approaches have utility in improving diagnostic testing and identifying causal haplotypes or SNPs.
PMCID: PMC3238183  PMID: 21884576
15.  Transancestral mapping of the MHC region in systemic lupus erythematosus identifies new independent and interacting loci at MSH5, HLA-DPB1 and HLA-G 
Annals of the Rheumatic Diseases  2012;71(5):777-784.
Systemic lupus erythematosus (SLE) is a chronic multisystem genetically complex autoimmune disease characterised by the production of autoantibodies to nuclear and cellular antigens, tissue inflammation and organ damage. Genome-wide association studies have shown that variants within the major histocompatibility complex (MHC) region on chromosome 6 confer the greatest genetic risk for SLE in European and Chinese populations. However, the causal variants remain elusive due to tight linkage disequilibrium across disease-associated MHC haplotypes, the highly polymorphic nature of many MHC genes and the heterogeneity of the SLE phenotype.
A high-density case-control single nucleotide polymorphism (SNP) study of the MHC region was undertaken in SLE cohorts of Spanish and Filipino ancestry using a custom Illumina chip in order to fine-map association signals in these haplotypically diverse populations. In addition, comparative analyses were performed between these two datasets and a northern European UK SLE cohort. A total of 1433 cases and 1458 matched controls were examined.
Using this transancestral SNP mapping approach, novel independent loci were identified within the MHC region in UK, Spanish and Filipino patients with SLE with some evidence of interaction. These loci include HLA-DPB1, HLA-G and MSH5 which are independent of each other and HLA-DRB1 alleles. Furthermore, the established SLE-associated HLA-DRB1*15 signal was refined to an interval encompassing HLA-DRB1 and HLA-DQA1. Increased frequencies of MHC region risk alleles and haplotypes were found in the Filipino population compared with Europeans, suggesting that the greater disease burden in non-European SLE may be due in part to this phenomenon.
These data highlight the usefulness of mapping disease susceptibility loci using a transancestral approach, particularly in a region as complex as the MHC, and offer a springboard for further fine-mapping, resequencing and transcriptomic analysis.
PMCID: PMC3329227  PMID: 22233601
16.  IGF-1, IGFBP-1, and IGFBP-3 Polymorphisms Predict Circulating IGF Levels but Not Breast Cancer Risk: Findings from the Breast and Prostate Cancer Cohort Consortium (BPC3) 
PLoS ONE  2008;3(7):e2578.
IGF-1 has been shown to promote proliferation of normal epithelial breast cells, and the IGF pathway has also been linked to mammary carcinogenesis in animal models. We comprehensively examined the association between common genetic variation in the IGF1, IGFBP1, and IGFBP3 genes in relation to circulating IGF-I and IGFBP-3 levels and breast cancer risk within the NCI Breast and Prostate Cancer Cohort Consortium (BPC3). This analysis included 6,912 breast cancer cases and 8,891 matched controls (n = 6,410 for circulating IGF-I and 6,275 for circulating IGFBP-3 analyses) comprised primarily of Caucasian women drawn from six large cohorts. Linkage disequilibrium and haplotype patterns were characterized in the regions surrounding IGF1 and the genes coding for two of its binding proteins, IGFBP1 and IGFBP3. In total, thirty haplotype-tagging single nucleotide polymorphisms (htSNP) were selected to provide high coverage of common haplotypes; the haplotype structure was defined across four haplotype blocks for IGF1 and three for IGFBP1 and IGFBP3. Specific IGF1 SNPs individually accounted for up to 5% change in circulating IGF-I levels and individual IGFBP3 SNPs were associated up to 12% change in circulating IGFBP-3 levels, but no associations were observed between these polymorphisms and breast cancer risk. Logistic regression analyses found no associations between breast cancer and any htSNPs or haplotypes in IGF1, IGFBP1, or IGFBP3. No effect modification was observed in analyses stratified by menopausal status, family history of breast cancer, body mass index, or postmenopausal hormone therapy, or for analyses stratified by stage at diagnosis or hormone receptor status. In summary, the impact of genetic variation in IGF1 and IGFBP3 on circulating IGF levels does not appear to substantially influence breast cancer risk substantially among primarily Caucasian postmenopausal women.
PMCID: PMC2440354  PMID: 18596909
17.  A cis-eQTL of HLA-DRB1 and a frameshift mutation of MICA contribute to the pattern of association of HLA alleles with cervical cancer 
Cancer Medicine  2014;3(2):445-452.
The association of classic human leukocyte antigen (HLA) alleles with risk of cervical cancer has been extensively studied, and a protective effect has consistently been found for DRB1*1301, DQA1*0103, and/or DQB1*0603 (these three alleles are in perfect linkage disequilibrium [LD] and often occur on the same haplotype in Europeans), while reports have differed widely with respect to the effect of HLA-B*07, DRB1*1501, and/or DQB1*0602 (the last two alleles are also in perfect LD in Europeans). It is not clear whether the reported HLA alleles are responsible for the differences in cervical cancer susceptibility, or if functional variants at other locations within the major histocompatibility complex (MHC) region may explain the effect. In order to assess the relative contribution of both classic HLA alleles and single-nucleotide polymorphisms (SNPs) within the MHC region to cervical cancer susceptibility, we have imputed classic HLA alleles in 1034 cervical cancer patients and 3948 controls in a Swedish population for an integrated analysis. We found that the protective haplotype DRB1*1301-DQA1*0103-DQB1*0603 has a direct effect on cervical cancer and always occurs together with the C allele of a HLA-DRB1 cis-eQTL (rs9272143), which increases the expression of HLA-DRB1. The haplotype rs9272143C-DRB1*1301-DQA1*0103-DQB1*0603 conferred the strongest protection against cervical cancer (odds ratio [OR] = 0.41, 95% confidence interval [CI] = 0.32–0.52, P = 6.2 × 10−13). On the other hand, the associations with HLA-B*0702 and DRB1*1501-DQB1*0602 are attributable to the joint effects of both the HLA-DRB1 cis-eQTL (rs9272143) and a frameshift mutation (G inserion of rs67841474, also known as A5.1) of the MHC class I polypeptide-related sequence A gene (MICA). Variation in LD between the classic HLA loci, rs9272143 and rs67841474 between populations may explain the different associations of HLA-B*07 and DRB1*1501-DQB1*0602 with cervical cancer between studies. The mechanism suggested may also explain similar inconsistent results for other HLA-associated diseases.
PMCID: PMC3987094  PMID: 24520070
Cervical cancer; cis-eQTL; frameshift mutation; HLA; MICA
18.  Association of ERAP1 Allelic Variants with Risk of Ankylosing Spondylitis  
Acta Naturae  2010;2(3):72-77.
Ankylosing spondylitis (AS) belongs to a group of autoimmune diseases affecting the axial skeleton. Beside thehla-b*27allele, several other human genes that control the variety processes of immune homeostasis are considered to be associated with AS manifestation in different human populations. Among strong associated non-MHC geneserap1 encodingthe endoplasmic reticulum aminopeptidase 1 isoform was recently identified by single nucleotide polymorphisms (SNPs) meta analysis. In our study we inspected the genetic association of five non-synonymous coding SNPs fromerap1 withAS in Caucasians. We implemented the SSP-PCR system for precise genotyping of 87hla-b*27positive AS patients and 77hla-b*27healthy donors from the Russian population. Considerable differences in allele’s frequencies within patients vs control cohort were shown for 3 of 5 SNPs under investigation. Using the EM-algorhitm we reconstructed 3-marker haplotypes that distinguish with high probability two cohorts due to differences in the haplotypes frequencies. In such a way both the sensitive, CCT, haplotype and the protective, TTC, one were predicted. To verify the calculation we determined genuine frequencies of 5-marker haplotypes in AS cohort by haplotyping of individual cDNA samples using improved SSP-PCR primer set. We demonstrated that the frequencies ofin silicareconstucted haplotypes and the frequencies of experimentally detected haplotypes are in a good agreement. Frequency of the risk haplotype CCT (rs17482078/10050860/2287987) detected within AS cohort reaches 88%, as well as the frequency calculated by EM-algorhitm.
PMCID: PMC3347560  PMID: 22649653
aminopeptidase ERAP1; human single nucleotide polymorphism; allele-specific PCR; human haplotypes; ankylosing spondylitis
19.  Peptidyl arginine deiminase type IV (PADI4) haplotypes interact with shared epitope regardless of anti-cyclic citrullinated peptide antibody or erosive joint status in rheumatoid arthritis: a case control study 
Arthritis Research & Therapy  2010;12(3):R115.
Anti-cyclic citrullinated peptide autoantibodies (anti-CCP) are the most specific serologic marker for rheumatoid arthritis (RA). Genetic polymorphisms in a citrullinating (or deiminating) enzyme, peptidyl arginine deiminase type IV (PADI4) have been reproducibly associated with RA susceptibility in several populations. We investigated whether PADI4 polymorphisms contribute to anti-CCP-negative as well as -positive RA, whether they influence disease severity (erosive joint status), and whether they interact with two major risk factors for RA, Human Leukocyte Antigen-DRB1 (HLA-DRB1) shared epitope (SE) alleles and smoking, depending on anti-CCP and erosive joint status.
All 2,317 unrelated Korean subjects including 1,313 patients with RA and 1,004 unaffected controls were genotyped for three nonsynonymous (padi4_89, padi4_90, and padi4_92) and one synonymous (padi4_104) single-nucleotide polymorphisms (SNPs) in PADI4 and for HLA-DRB1 by direct DNA sequence analysis. Odds ratios (OR) were calculated by multivariate logistic regression. Interaction was evaluated by attributable proportions (AP), with 95% confidence intervals (CI).
A functional haplotype of the three fully correlated nonsynonymous SNPs in PADI4 was significantly associated with susceptibility to not only anti-CCP-positive (adjusted OR 1.73, 95% CI 1.34 to 2.23) but also -negative RA (adjusted OR 1.75, 95% CI 1.15 to 2.68). A strong association with both non-erosive (adjusted OR 1.62, 95% CI 1.29 to 2.05) and erosive RA (adjusted OR 1.62, 95% CI 1.14 to 2.31) was observed for PADI4 haplotype. Gene-gene interactions between the homozygous RA-risk PADI4 haplotype and SE alleles were significant in both anti-CCP-positive (AP 0.45, 95% CI 0.20 to 0.71) and -negative RA (AP 0.61, 95% CI 0.29 to 0.92). Theses interactions were also observed for both non-erosive (AP 0.48, 95% CI 0.25 to 0.72) and erosive RA (AP 0.46, 95% CI 0.14 to 0.78). In contrast, no interaction was observed between smoking and PADI4 polymorphisms.
A haplotype of nonsynonymous SNPs in PADI4 contributes to development of RA regardless of anti-CCP or erosive joint status. The homozygous PADI4 haplotype contribution is affected by gene-gene interactions with HLA-DRB1 SE alleles.
PMCID: PMC2911908  PMID: 20537173
20.  Association Assessment of Copy Number Polymorphism and Risk of Age-Related Macular Degeneration 
Ophthalmology  2011;118(12):2442-2446.
We previously identified a genetic copy number polymorphism (CNP147) that was statistically associated with age-related macular degeneration (AMD), and which resides downstream of the complement factor H (CFH) gene. Factor H protein is polymorphic at amino acid 402 in which the resulting histidine containing moiety has been established to impart significant risk of AMD. Here we present a method to precisely determine the exact copy number of CNP147 and examine in more detail the association with AMD.
Case-control Study
421 AREDS (Age-related Eye Disease cohort Study) subjects of whom approximately 35% were diagnosed with neovascular disease, 19% with geographic atrophy, 16% with both, 30% with large drusen and 215 controls.
Using copy number assays available from Applied Biosystems Inc., we examined four loci spanning CNP147 and neighboring CNP148 in an AREDS matched case-control sample set. We analyzed these data by copy number while controlling for two high-risk CFH variants, rs1061170 (Y402H) and rs1410996. We phased the high risk CFH variants with CNP147 and analyzed haplotype frequencies in cases and controls. To further validate copy numbers, six Utah CEPH families (Centre D’etude du Polymorphism Humaine) were typed for CNP147 and the segregation assessed.
Main Outcome Measures
Increased or decreased risk of AMD from genetic loci.
Having fewer than 2 copies of CNP147 is associated with an estimated 43% reduction in odds of having AMD in this sample set (adjusted odds ratio=0.57, P=0.006). CNP148 variation is rare in Caucasians and it was not statistically significant. Common haplotypes reveal that the risk alleles for rs1061170 and rs1410996 most frequently segregate with higher copy numbers for CNP147; but not exclusively, and that one haplotype that carried a deletion of CNP147 was highly protective (odds ratio=0.25 P=1.3×10−13) when compared to the reference.
In this matched subset of AREDS subjects, after adjusting for two known risk variants in CFH, CNP147 deletion statistically associates with diminished risk for AMD.
PMCID: PMC3223559  PMID: 21856016
Copy number polymorphism; age-related macular degeneration; HapMap 3; TaqMan Copy Number Assays; qPCR
21.  Dominant Sequences of Human Major Histocompatibility Complex Conserved Extended Haplotypes from HLA-DQA2 to DAXX 
PLoS Genetics  2014;10(10):e1004637.
We resequenced and phased 27 kb of DNA within 580 kb of the MHC class II region in 158 population chromosomes, most of which were conserved extended haplotypes (CEHs) of European descent or contained their centromeric fragments. We determined the single nucleotide polymorphism and deletion-insertion polymorphism alleles of the dominant sequences from HLA-DQA2 to DAXX for these CEHs. Nine of 13 CEHs remained sufficiently intact to possess a dominant sequence extending at least to DAXX, 230 kb centromeric to HLA-DPB1. We identified the regions centromeric to HLA-DQB1 within which single instances of eight “common” European MHC haplotypes previously sequenced by the MHC Haplotype Project (MHP) were representative of those dominant CEH sequences. Only two MHP haplotypes had a dominant CEH sequence throughout the centromeric and extended class II region and one MHP haplotype did not represent a known European CEH anywhere in the region. We identified the centromeric recombination transition points of other MHP sequences from CEH representation to non-representation. Several CEH pairs or groups shared sequence identity in small blocks but had significantly different (although still conserved for each separate CEH) sequences in surrounding regions. These patterns partly explain strong calculated linkage disequilibrium over only short (tens to hundreds of kilobases) distances in the context of a finite number of observed megabase-length CEHs comprising half a population's haplotypes. Our results provide a clearer picture of European CEH class II allelic structure and population haplotype architecture, improved regional CEH markers, and raise questions concerning regional recombination hotspots.
Author Summary
The human major histocompatibility complex (MHC) is a gene-dense region highly enriched in immune response genes. MHC genetic variation is among the highest in the human genome and is associated with both tissue transplant compatibility and many genetic disorders. Long-range (1–3 Mb) MHC haplotypes of essentially identical DNA sequence at relatively high (≥0.5%) population frequency (“genetic fixity”), called conserved extended haplotypes (CEHs), comprise roughly half of all European population haplotypes. We sequenced an aggregate of 27 kb over 580 kb in the MHC class II region from HLA-DQA2 to DAXX in 158 European haplotypes to quantify the breakdown of this genetic fixity in the centromeric portion of the MHC and to determine the representative nature within that region of eight previously fully or nearly fully sequenced “common” European haplotypes. We identified the dominant sequences of 13 European CEHs and determined where the “common” sequences did (or did not) represent related CEHs. We found patterns of shared sequence identity among different CEHs surrounded by fixed (for each CEH) but differing sequence. Our direct observational results for population haplotypes explain the mutual occurrence of CEHs and short (5–200 kb) blocks of fixed sequence detected by the statistical measure of linkage disequilibrium.
PMCID: PMC4191933  PMID: 25299700
22.  Identification of HLA-A*02-B*46 haplotype allele variant in Guangdong Han populations on the basis of PCR-SBT 
BMC Research Notes  2009;2:55.
The HLA-A*02-B*46 haplotype is one of most frequent haplotypes among Guangdong Han populations. To explore the characteristics of the HLA-A*02-B*46 haplotype in Guangdong Han populations, the genetic polymorphism of HLA-A*02-B*46 haplotype was analysed by PCR-SBT in our study.
Among 88 samples with the homozygotes for HLA-A*02-B*46 in the low resolution, 4 different alleles for A*02 (A*0201, A*0203, A*0206, A*0207) and 1 allele for B*46 (B*4601) were identified by PCR-SBT. Among them, the A*0207 allele was the predominant allele. Inversely, among the samples with HLA-A*2-B*46(-), six alleles were detected for A*02 (A*020101, A*0203, A*0205, A*0206 and A*0207), and the A*0201 allele was predominant. On the other hand, the HLA-A*02-B*46 haplotype presented moderate heterozygosis (32.95%). In addition, the linkage with DRB1 was analysed in HLA-A*2-B*46 haplotype, and there existed 10 alleles with DRB1. With the low resolution for DRB1, the other 10 DRB1 alleles all linked with the HLA-A*02-B*46 haplotype except for DRB1*01, DRB1*10, and DRB1*17. Moreover, we found eight alleles of DRB1 in the HLA-A*0207-B*4601 haplotype.
The polymorphism distribution of the A*02 allele between the HLA-A*02-B*46 and HLA-A*02-B*46(-) haplotypes among the Guangdong Han populations provides useful information for research on unrelated hematopoietic stem cell transplantation (UHSCT), anthropology, and disease association for populations with the HLA-A*02-B*46 haplotype.
PMCID: PMC2674454  PMID: 19348686
23.  Diversity of Extended HLA-DRB1 Haplotypes in the Finnish Population 
PLoS ONE  2013;8(11):e79690.
The Major Histocompatibility Complex (MHC, 6p21) codes for traditional HLA and other host response related genes. The polymorphic HLA-DRB1 gene in MHC Class II has been associated with several complex diseases. In this study we focus on MHC haplotype structures in the Finnish population. We explore the variability of extended HLA-DRB1 haplotypes in relation to the other traditional HLA genes and a selected group of MHC class III genes. A total of 150 healthy Finnish individuals were included in the study. Subjects were genotyped for HLA alleles (HLA-A, -B, -DRB1, -DQB1, and -DPB1). The polymorphism of TNF, LTA, C4, BTNL2 and HLA-DRA genes was studied with 74 SNPs (single nucleotide polymorphism). The C4A and C4B gene copy numbers and a 2-bp silencing insertion at exon 29 in C4A gene were analysed with quantitative genomic realtime-PCR. The allele frequencies for each locus were calculated and haplotypes were constructed using both the traditional HLA alleles and SNP blocks. The most frequent Finnish A∼B∼DR -haplotype, uncommon in elsewhere in Europe, was A*03∼B*35∼DRB1*01∶01. The second most common haplotype was a common European ancestral haplotype AH 8.1 (A*01∼B*08∼DRB1*03∶01). Extended haplotypes containing HLA-B, TNF block, C4 and HLA-DPB1 strongly increased the number of HLA-DRB1 haplotypes showing variability in the extended HLA-DRB1 haplotype structures. On the contrary, BTNL2 block and HLA-DQB1 were more conserved showing linkage with the HLA-DRB1 alleles. We show that the use of HLA-DRB1 haplotypes rather than single HLA-DRB1 alleles is advantageous when studying the polymorphisms and LD patters of the MHC region. For disease association studies the HLA-DRB1 haplotypes with various MHC markers allows us to cluster haplotypes with functionally important gene variants such as C4 deficiency and cytokines TNF and LTA, and provides hypotheses for further assessment. Our study corroborates the importance of studying population-specific MHC haplotypes.
PMCID: PMC3836878  PMID: 24278156
24.  The -1997 G/T and Sp1 Polymorphisms in the Collagen Type I alpha1 (COLIA1) Gene in Relation to Changes in Femoral Neck Bone Mineral Density and the Risk of Fracture in the Elderly: The Rotterdam Study 
Calcified Tissue International  2007;81(1):18-25.
The COLIA1 Sp1 polymorphism has been associated with bone mineral density (BMD) and fracture. A promoter polymorphism, -1997 G/T, also has been associated with BMD. In this study, we examined whether these polymorphisms alone and in the form of haplotypes influence bone parameters and fracture risk in a large population-based cohort of elderly Caucasians. We determined the COLIA1 -1997 G/T (promoter) and Sp1 G/T (intron) polymorphisms in 6,280 individuals and inferred haplotypes. Femoral neck BMD and BMD change were compared across COLIA1 genotypes at baseline and follow-up (mean 6.5 years). We also investigated the relationship between the COLIA1 polymorphisms and incident nonvertebral fractures, which were recorded during a mean follow-up period of 7.4 years. Vertebral fractures were assessed by radiographs on 3,456 genotyped individuals. Femoral neck BMD measured at baseline was 3.8% lower in women carrying two copies of the T-Sp1 allele (P for trend = 0.03). No genotype dependent differences in BMD loss were observed. In women homozygous for the T allele of the Sp1 polymorphism, the risk of fragility fracture increased 2.3 times (95% confidence interval 1.4–3.9, P = 0.001). No such association was observed with the promoter polymorphism. In men, no association with either the Sp1 or the -1997 G/T promoter polymorphism was seen with BMD or fracture. High linkage disequilibrium (LD; D′ = 0.99, r2 = 0.03) exists between the two studied polymorphisms. We observed three haplotypes in our population: haplotype 1 (Gpromoter–Gintron) frequency (f) = 69%, haplotype 2 (Gpromoter–Tintron) f = 17.6%, and haplotype 3 (Tpromoter–Gintron) f = 13.4%. Haplotype 2 was associated with a 2.1-fold increased risk of fragility fracture in women (95% confidence interval 1.2–3.7, P = 0.001). We confirm that the COLIA1 Sp1 polymorphism influences BMD and the risk of fracture in postmenopausal Caucasian women. In contrast, we found no independent effect of the -1997 G/T promoter polymorphism on BMD or fracture.
PMCID: PMC1914224  PMID: 17551768
Bone; Osteoporosis; Bone mineral density; Fracture; COLIA1
25.  Genetic fixity in the human major histocompatibility complex and block size diversity in the class I region including HLA-E 
BMC Genetics  2007;8:14.
The definition of human MHC class I haplotypes through association of HLA-A, HLA-Cw and HLA-B has been used to analyze ethnicity, population migrations and disease association.
Here, we present HLA-E allele haplotype association and population linkage disequilibrium (LD) analysis within the ~1.3 Mb bounded by HLA-B/Cw and HLA-A to increase the resolution of identified class I haplotypes. Through local breakdown of LD, we inferred ancestral recombination points both upstream and downstream of HLA-E contributing to alternative block structures within previously identified haplotypes. Through single nucleotide polymorphism (SNP) analysis of the MHC region, we also confirmed the essential genetic fixity, previously inferred by MHC allele analysis, of three conserved extended haplotypes (CEHs), and we demonstrated that commercially-available SNP analysis can be used in the MHC to help define CEHs and CEH fragments.
We conclude that to generate high-resolution maps for relating MHC haplotypes to disease susceptibility, both SNP and MHC allele analysis must be conducted as complementary techniques.
PMCID: PMC1853106  PMID: 17430593

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